Method of lipid assay and reagent for use therein

ABSTRACT

A method of lipid assay characterized by assaying the lipids contained in a blood component in the presence of an organic silicon compound. The method can cause specific conditions for direct methods while satisfying requirements such as no influence on precision of assay, no burden on assay apparatus, and easy availability.

FIELD OF THE INVENTION

The present invention relates to a method of separately assaying lipidcomponents in specific lipoprotein fractions efficiently by a simpleprocedure using a small amount of sample. The present invention alsorelates to a reagent used for the method.

DESCRIPTION OF BACKGROUND ART

Cholesterols, triglycerides, and phospholipids which are major lipids inthe living body are combined with apoproteins to form lipoproteins inblood. The lipoproteins are grouped into chylomicron, very low densitylipoprotein (VLDL), low density lipoprotein (LDL), high densitylipoprotein (HDL), and the like according to their physical properties.Among these lipoproteins, it is known that LDL is a causative factor forarteriosclerosis, whereas HDL exhibits an anti-arteriosclerosis effect.

Total cholesterols and total triglycerides in blood have been assayedfor the purpose of preventing ischemic heart diseases or evaluating theeffects of treatments for such diseases. Epidemiological studies haveproved that the cholesterol level in LDL has a positive correlation withthe frequency of arteriosclerosis occurrence, whereas the cholesterollevel in HDL has a negative correlation with the frequency ofarteriosclerosis occurrence. Importance of knowing fluctuations in eachlipoprotein fraction such as LDL and HDL has been understood. Nowadays,apoprotein B and apoprotein A-I, which are protein components formingeach lipoprotein, or cholesterol in each lipoprotein fraction isassayed.

As mentioned above, as the method of assaying lipids components inblood, a method of determining the total amount of specific lipidcontained in all lipoproteins such as total cholesterol and totaltriglycerides and a method of determining cholesterols in specificlipoproteins such as LDL, HDL, and the like can be given.

Of these, the former method is based on the combination of an enzyme forthe lipid component to be assayed and conditions enabling the reactionof the lipid component with the enzyme without regard to the type oflipoproteins (for example, a surfactant with low lipoprotein selectivitysuch as Triton X-100). The method enables assaying the lipid componentsto be assayed in all lipoproteins.

The latter method, on the other hand, is based on the combination of anenzyme for the lipid component to be assayed and specific conditionsenabling the enzyme to react only with the lipid component to be assayedin specific lipoproteins. The method enables the assay of the lipidcomponents to be assayed in the specific lipoprotein fractions.

These specific conditions have been conventionally established by amethod of isolating the lipoproteins to be assayed from a sample byultracentrifugation, electrophoresis, gel filtration, precipitationusing a precipitation agent (precipitation method), or the like (themethod is hereinafter referred to as “fractionation method”).

In recent years, a number of methods for establishing specificconditions without using the fractionation method (hereinafter referredto as “direct methods”) have been developed. Such methods include amethod of utilizing the different reaction times of bile acid for eachlipoprotein fraction in the determination of HDL cholesterols (JapanesePatent Publication No. 016720/1994), a method of reacting thelipoproteins to be assayed with an enzyme under the condition thatlipoproteins not to be assayed stay aggregated (Japanese PatentApplication Laid-open Publication No. 242110/1994), a method of using asugar compound (Japanese Patent No. 2653755), a method of using amodification enzyme (Japanese Patent No. 2600065), a method of using asurfactant which does not dissolve lipoproteins (Japanese Patent No.2799835), a method of removing cholesterols in lipoproteins not to beassayed by a previous enzyme reaction (Japanese Patent ApplicationLaid-open Publication No. 000299/1997), a method of using carrageenan(Japanese Patent Application Laid-open Publication No. 121895/1997), amethod of using a surfactant having lipoprotein selectivity (JapanesePatent Application Laid-open Publication No. 056395/1999), a method ofusing Calixarene (WO 98/59068), and a method of using a phosphoruscompound (Japanese Patent Application Laid-open Publication No.116400/2000).

Some of these direct methods are used in practice in daily clinicaltests due to their simple procedures as compared with the precipitationmethod involving complicated procedures. However, even the directmethods which have been put into practice have problems. For example,the method of adding a precipitation agent to the reagent has problems:interference of the aggregates on assay precision and a damage tomeasuring equipment such as clogged flow passages with the productsproduced from reaction of the precipitation agent with an alkalinedetergent for washing measuring equipment. As for a method of using amodification enzyme, there are problems such as process control inenzyme modification procedures (quality control) and cost increase.

Accordingly, an object of the present invention is to provide a methodof assaying lipid components in blood and the like using a substancecapable of establishing the specific conditions under the direct methodwhile neither affecting assay precision nor damaging assay instruments,and also satisfying other conditions such as easy availability.

DISCLOSURE OF THE INVENTION

The inventors of the present invention have conducted extensive studiesto achieve the above object and completed the present invention with thefollowing method. As for assaying the lipid components in specificlipoproteins to be assayed such as HDL cholesterols, the HDLcholesterols alone can be selectively assayed with specificity even inthe absence of precipitation agents and modification enzymes if anorganic silicon compound is added to the reagent, and the enzyme havingthe cholesterol as a substrate is mixed with the sample to be assayed.

Specifically, the present invention provides a method of lipid assaycharacterized by the assay conducted in the presence of an organicsilicon compound and a reagent used for the assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart for showing the correlation between the results ofassaying HDL cholesterol in a blood serum sample obtained by the methodof the present invention shown in Example 2 or the control method usinga reagent not containing an organic silicon compound of the presentinvention and the results obtained by the precipitation method, whereinA shows the correlation for the control method and B shows thecorrelation for the method of the present invention.

FIG. 2 is a chart for showing the correlation between the results ofassaying HDL cholesterol in a blood serum sample obtained by the methodof the present invention shown in Example 3A or 3B or the control methodusing a reagent not containing the organic silicon compound of thepresent invention and the results obtained by the precipitation method,wherein A, B, and C show the correlations for the control method, themethod of Example 3A, and the method of Example 3B respectively.

FIG. 3 is a chart for showing the correlation between the results ofassaying HDL cholesterol in a blood serum sample obtained by the methodof the present invention shown in Example 4 or the control method usinga reagent not containing an organic silicon compound of the presentinvention and the results obtained by the precipitation method, whereinA shows the correlation for the control method and B shows thecorrelation for the method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The method of the present invention comprises assaying lipid using aknown reagent for assaying lipids in the presence of an organic siliconcompound.

Silicones and their derivatives can be given as the organic siliconcompound in the present invention. The silicones are the polymers whichhave organic groups such as an alkyl group or aryl group. The polymerscomprise —(Si—O)_(n)—, which is generally called polysiloxane, therecurrence ⁶structure of siloxane unit which consists of a silicon atombonded with an oxygen atom. Properties of silicones differ according tothe molecular structure. Viscosity, volatility, and other propertiesvary according to the degree of polymerization, types of side chains,the degree of crosslinking, and the like. There are liquid silicones,greasy silicones, rubbery silicones, and resinous silicones. Thesesilicones and their derivatives are commercially available underclassification names of silicone, high polymerization silicone, cyclicsilicone, alkyl silicone, silicone-containing surfactant, modifiedsilicone oil, and the like. The modified silicone oil is the siliconeoil to which an organic group is added. According to the added organicgroup, the modified silicone oil is classified as an amino modifiedsilicone oil, epoxy modified silicone oil, carboxyl modified siliconeoil, polyether modified silicone oil, alkyl modified silicone oil, orthe like. According to the structure, the modified silicone oil isclassified as a side chain silicone oil, both terminal silicone oil, oneterminal silicone oil, and side chain both terminal silicone oil. Thesesilicones and derivatives may be used either individually or incombination of two or more in achieving the object of the presentinvention. Manufacturers publish catalogues describing properties andthe like of their organic silicon products. Specific organic siliconcompounds to be used may be selected by referring to these catalogues.

In measuring the target lipid, the organic silicon compound of thepresent invention may be either added simultaneously with the sample andthe reagent for assaying the target lipid or mixed with the samplebefore adding the reagent for assaying the target lipid. Alternatively,the target lipid may be assayed by first preparing a mixture of thesample and a part of the reagent for assaying the target lipid andadding to the mixture the remaining portion of the reagent containingthe organic silicon compound of the present invention.

There are no specific limitations to the method of detecting the targetlipid after the addition of the reagent for assaying the target lipid.For example, spectrophotometric analysis using a combination of aperoxidase and a chromogen and a method of directly detecting a coenzymeand hydrogen peroxide can be given.

The amount of the organic silicon compound used for the sample in thepresent invention varies according to the type of the lipid to beassayed, the properties of the sample, the type of the reagent used, thepresence or absence of the later-described surfactant or glycerol, orthe amount of the surfactant or glycerol, and the like. Although optimumconditions should be selected by experiment, generally used amount ofthe organic silicon compound is about 0.0001-5 mass % (hereinaftersimply referred to as “%”), and more preferably about 0.001-5%.

It is preferable to apply the above-described direct method to thepresent invention, that is, it is preferable to use the organic siliconcompound of the present invention in combination with the reagent forthe direct method. When used in combination with a surfactant which doesnot dissolve lipoproteins (Japanese Patent No. 2799835) or a surfactanthaving lipoprotein selectivity (Japanese Patent Application Laid-openPublication No. 056395/1999), for example, the organic silicon compoundof the present invention can enhance the properties of thesesurfactants.

In addition, it is possible to emulsify the organic silicon compound ofthe present invention by adding a surfactant, glycerol, and the like toadjust the water solubility.

As the surfactant, any of anionic surfactants, nonionic surfactants,cationic surfactants, and ampholytic surfactants may be used withoutspecific limitations. Examples include alkyl ether carboxylates, saltsof N-acylamino acid, alkyl phosphates, N-acyl taurates, sulfonates,alkyl sulfates, polyoxyethylene alkyl ether sulfates, acetic acidbetaines, imidazolines, alkyl ammonium salts, amide amines, polyhydricalcohol fatty acid esters, alkyl glyceryl ethers and their fatty acidesters, propylene glycol fatty acid esters, glycerides, polyglycerides,polyoxyethylene glycerides, sorbitan fatty acid esters, polyoxysorbitanfatty acid esters, polyoxyethylene sorbit fatty acid esters,polyoxyethylene lanolins, polyoxyethylene lanolin alcohols,polyoxyethylene bees wax derivatives, polyoxyethylene castor oil,polyoxyethylene hydrogenated castor oil, polyoxyethylene sterols,polyoxyethylene hydrogenated sterols, polyethylene glycol fatty acidesters, polyoxyethylene alkyl ethers, polyoxyethylene polyoxypropylenealkyl ethers, polyoxyethylene polyoxypropylene condensates ofethylenediamine, polyoxyethylene alkylphenyl ethers, polyoxyethylenealkyl amines, sucrose fatty acid esters, polyoxyethylene fatty acidamides, polyoxyethylene alkylphenyl formaldehyde condensates,polyoxyethylene alkyl ether phosphoric acid, salts of polyoxyethylenealkyl ether phosphoric acid, and the like.

These surfactants may be added to the assay system either individuallyor in combination of two or more. The combination and amount of thesesurfactants vary according to the type of silicones and theirderivatives, the type of surfactant used, and the like. Although optimumconditions should be selected by experiment, generally usedconcentration of the surfactant in the assay system is about 0.0001-5%,and more preferably about 0.001-5%.

In addition to the surfactant, polymer emulsifying agents such as anacrylic acid-alkyl methacrylate copolymer and carboxy vinyl polymer,organic solvents such as ethanol, silicones and their derivatives notadversely affecting the assay specificity, and the like can be used inadjusting the water solubility.

The lipid to be assayed by the method of the present invention may beany lipid present in the living body, and particularly preferable lipidsare those forming lipoproteins such as cholesterols, triglycerides, andphospholipids.

Any enzyme having the target lipid as the substrate can be used inassaying the lipid in the method of the present invention. Enzymescommonly used for assaying cholesterols, triglycerides, andphospholipids described in Outline of Clinical Diagnosis Method, the30^(th) edition, (Kanehara and Co., Ltd., 1993), for example, can beused in the present invention and are included in the scope of thepresent invention.

When cholesterols are assayed among these lipids, enzymes commonly usedfor assaying cholesterols such as cholesterol dehydrogenase, cholesteroloxidase, and cholesterol esterase can be used without a limitation.These enzymes may be those originating from microorganisms, animals, orplants or may be those prepared by a genetic engineering technique.Either chemically modified or unmodified enzymes can be used.

These enzymes may be optionally used in combination with a coenzyme, adetecting enzyme or dyeing agent, or the like. As the coenzyme, nicotineamide adenine dinucleotide and the like can be given. As the detectingenzyme, peroxidase and the like can be given. As the dyeing agent,Trinder reagent, aminoantipyrine, and the like can be given.

The detecting enzymes may be used either individually or in combinationof two or more. The amount used differs according to the enzymes.Although not specifically limited, the enzyme in the amount of 0.001-100unit/mL, and preferably 0.1-100 unit/mL, is used.

The method of the present invention can be advantageously carried out byusing a reagent for assaying lipid prepared by appropriately formulatingthe above-described organic silicon compound, enzyme for assaying lipid,dyeing agent, coenzyme, and the like. As required, reagents havingaffinity with specific lipoproteins, including other enzymes such ascatalase, salts, pH adjusting buffering agents, surfactants, antisepticagents, proteins such as albumin, antibodies, antibiotics, saponins,lectins, and polyanions, can be added to the extent that the assayspecificity is not adversely affected and the specific lipoproteins arenot aggregated.

As the buffering agent, any buffering agent such as Good's buffer,phosphoric acid, Tris buffer, and phthalate may be used inasmuch as thebuffering agent can establish buffering conditions of pH of the reactionsolution in the range of 4-10. Although not specifically limited, theamount in the range of 0.0005-2 mol/L, and preferably of 0.01-1 mol/L,is applicable. In practice, optimum conditions should be selected byexperiment in due consideration to the properties of the enzyme used,other components contained in the reagent, and the like.

Specific examples of the reagent for assaying lipid for advantageouslycarrying out the method of the present invention are given below.

(HDL-Cholesterol Assay Reagent)

(1) Organic silicon compound

(2) Cholesterol esterase

(3) Cholesterol oxidase

(4) Peroxidase

(5) Dyeing agent (4-aminoantipyrine and disulfobutyl metatoluidine,etc.)

-   -   A surfactant having lipoprotein selectivity and other additives        may be optionally added.

(LDL-Cholesterol Assay Reagent) (First Reagent)

(1) Organic silicon compound

(2) Cholesterol esterase

(3) Cholesterol oxidase

(4) Peroxidase

(5) Either one of two compounds generating a color by combination (e.g.4-aminoantipyrine)

-   -   A surfactant having lipoprotein selectivity and other additives        may be optionally added.

(Second Reagent)

(6) The other of two compounds generating a color by combination (e.g.disulfobutylmetatoluidine)

(7) Surfactant with low lipoprotein selectivity

EXAMPLES

The present invention will be described in more detail by way ofExamples which should not be construed as limiting the presentinvention.

Example 1

The effect of the present invention was confirmed using HDL and LDLfractions prepared by ultracentrifugation as the samples. The reagentwith the following formulation was used for assaying cholesterols in theHDL and LDL fractions.

(First reagent) PIPES buffer solution (pH 6.5) 50 mmol/L4-Aminoantipyrine 0.5 mmol/L (Second reagent) PIPES buffer solution (pH6.5) 50 mmol/L Cholesterol esterase 1 unit/mL Cholesterol oxidase 1unit/mL Disulfobutylmetatoluidine 1.0 mmol/L Peroxidase 5 unit/mLOrganic silicon compound 1% (all manufactured by Nippon Unicar Co.,Ltd.)

Hitachi 7170 automatic analyzer was used. 240 μL of the first reagentwas added to 2.4 μL of the sample. Five minutes later, 80 μL of thesecond reagent was added. Absorbance at a wavelength of 600 nm wasmeasured immediately before the addition of the second reagent and 5minutes thereafter. The difference of the two measurements was regardedas the absorbance of the sample. As a control, a reagent which contains1% Triton X-100 having low lipoprotein selectivity instead of theorganic silicon compound of the present invention was used as the secondreagent.

Relative sample absorbances measured using reagents containing adifferent organic silicon compound for each as the second reagents weredetermined assuming the absorbance of the sample using the secondreagent containing the Triton X-100 as 100. The ratio of the relativesample absorbance for LDL to the relative sample absorbance for HDL wascalculated. The results are shown in Table 1.

TABLE 1 Relative Relative Ratio of relative absorbance absorbanceabsorbance Added components for HDL for LDL (LDL/HDL) Organic silicon 231 0.04 compound 1 (NUC-Silicon L7002) Organic silicon 94 9 0.10 compound2 (NUC-Silicon L7604) Organic silicon 49 15 0.31 compound 3 (NUC-SiliconFZ2118) Organic silicon 14 7 0.50 compound 4 (NUC-Silicon FZ2162)Organic silicon 49 16 0.33 compound 5 (NUC-Silicon FZ2163) Surfactant(Triton X-100) 100 100 1.00

The LDL/HDL ratio of the relative absorbance when measured by using thesecond reagents containing the organic silicon compounds of the presentinvention was in the range of 0.04-0.5. The results indicate that theorganic silicon compound of the present invention established specialconditions under which the reaction between cholesterols in HDL andenzymes is predominant over the reaction between cholesterols in LDL andenzymes.

Example 2

The effect of the present invention was confirmed using 20 serum samplescontaining lipoproteins. Reagents with the following formulation wereused for assaying.

(First reagent) Bis-Tris buffer solution (pH 6.0) 50 mmol/L Cholesteroloxidase 1 unit/mL Peroxidase 1.25 unit/mL Disulfobutylmetatoluidine 0.5mmol/L Flufenamic acid 150 μmol/L (Second reagent) Bis-Tris buffersolution (pH 6) 50 mmol/L Cholesterol esterase 1.5 unit/mL (Asahi KaseiCorporation) 4-Aminoantipyrine 1.0 mmol/L Emulgen B-66 1.5% Organicsilicon compound 0.001% (NUC-Silicon L720 manufactured by Nippon UnicarCo., Ltd.)

Hitachi 7170 automatic analyzer was used. 240 μL of the first reagentwas added to 2.4 μL of the sample. Five minutes later, 80 μL of thesecond reagent was added. Absorbance at 600 nm wavelength was measuredimmediately before the addition of the second reagent and 5 minutesafter the addition of the second reagent, to determine the HDLcholesterol concentration from the difference in the absorbance values(Two point method). A control serum with a known concentration was usedas a calibration substance. As a control, a reagent not containing theorganic silicon compound of the present invention was used as the secondreagent.

At the same time, the HDL cholesterol concentration in the serum samplewas determined using a commercially available precipitation reagent kit(HDL-C•2 kit manufactured by Daiichi Pure Chemicals Co., Ltd.). Thecorrelation coefficients and regression formula were compared, takingthe value of the reagent in the precipitation method as X axis and themeasured value in Examples as Y axis. The results are shown in Table 2and FIG. 1.

TABLE 2 Unit: mg/dL Precipitation Method of Sample method Control thepresent invention 1 83.2 81.4 84.4 2 83.2 75.2 77.4 3 72.7 70.4 71.3 469.8 64.0 64.7 5 68.1 67.6 68.3 6 68.3 65.5 66.3 7 62.3 60.0 60.8 8 61.159.0 59.1 9 56.9 56.2 56.5 10 54.4 49.3 49.2 11 52.7 51.8 53.2 12 52.352.5 52.6 13 49.0 48.0 48.4 14 45.6 48.1 48.1 15 43.3 40.9 41.1 16 39.839.8 39.9 17 39.5 41.3 40.7 18 32.3 33.0 32.7 19 32.2 33.8 33.5 20 66.968.2 68.3 Correlation coefficient — 0.989 0.989 Slope — 0.891 0.935Intercept — 4.8 2.8

The results measured by using the second reagent containing the organicsilicon compound of the present invention exhibited improved slope andintercept as compared with the results measured by using the secondreagent not containing the organic silicon compound of the presentinvention. This indicates that the organic silicon compound of thepresent invention established specific conditions under which thereaction between cholesterols in HDL and enzymes is predominant over thereaction between cholesterols in LDL and enzymes, whereby thecharacteristics of the surfactant having lipoprotein selectivity wasfurther improved.

Example 3

The effect of the present invention was confirmed in the same conditionas in Example 2 except that 26 serum samples containing lipoproteinswere used as the samples and NET-SG-60A (Example 3A) or NET-SG-60C(Example 3B) (both at a concentration of 0.05%, manufactured by NihonSurfactant Kogyo KK.) was used as the organic silicon compound of thepresent invention in the second reagent. The results are shown in Table3 and FIG. 2.

TABLE 3 Unit: mg/dL Precipitation Method of the present invention Samplemethod Control Example 3A Example 3B 1 44.5 47.8 46.7 47.0 2 49.3 51.150.9 51.1 3 48.8 51.1 51.0 50.9 4 69.7 72.4 72.7 73.6 5 67.2 66.9 67.967.5 6 66.4 69.1 69.1 69.1 7 40.3 42.2 42.2 42.2 8 28.6 32.4 30.8 30.5 945.3 47.9 46.6 46.6 10 46.8 48.2 48.1 48.2 11 41.7 44.1 43.3 43.0 1241.1 45.0 42.7 43.6 13 55.6 59.2 58.6 59.2 14 52.6 56.2 55.3 56.1 1562.2 62.2 63.0 62.3 16 32.7 35.9 34.8 35.0 17 48.2 48.3 48.5 49.2 1847.4 49.2 49.6 49.0 19 41.1 45.1 45.0 45.0 20 53.3 54.8 54.2 54.3 2144.0 46.0 45.8 45.9 22 75.3 78.2 79.5 80.1 23 59.4 61.7 60.5 61.2 2437.1 39.7 39.3 39.6 25 50.6 55.0 53.0 53.5 26 36.9 40.9 40.4 40.3Correlation — 0.995 0.997 0.996 coefficient Slope — 0.963 1.006 1.015Intercept — 4.3 1.8 1.5

The results measured by using the second reagent containing the organicsilicon compound of the present invention exhibited improved slope andintercept as compared with the results measured by using the secondreagent not containing the organic silicon compound of the presentinvention. This indicates that the organic silicon compound of thepresent invention established specific conditions under which thereaction between cholesterols in HDL and enzymes is predominant over thereaction between cholesterols in LDL and enzymes, whereby thecharacteristics of the surfactant having lipoprotein selectivity wasfurther improved.

Example 4

The effect of the present invention was confirmed in the same conditionas in Example 2 except that 30 serum samples containing lipoproteinswere used as the samples and KF-700 (0.08%, manufactured by Shin-EtsuChemical Co., Ltd.) was used as the organic silicon compound of thepresent invention in the second reagent. The results are shown in Table4 and FIG. 3.

TABLE 4 Precipitation Method of Sample method Control the presentinvention 1 98.6 98.7 98.2 2 95.0 94.5 95.0 3 87.4 87.9 89.4 4 84.0 84.784.5 5 80.4 82.6 81.8 6 76.6 79.0 79.2 7 74.2 76.6 76.1 8 71.8 74.6 74.19 69.2 70.6 70.0 10 63.6 67.6 67.4 11 60.0 63.8 64.2 12 60.8 64.7 63.913 59.8 63.2 62.9 14 58.6 60.9 61.3 15 55.8 59.7 59.2 16 55.8 60.0 59.117 54.4 58.3 57.1 18 52.0 56.3 54.6 19 49.0 53.8 52.8 20 48.6 49.5 48.821 45.0 48.2 47.5 22 43.4 47.1 46.6 23 45.0 46.8 46.0 24 44.6 47.0 45.825 42.0 44.0 42.9 26 41.8 43.0 42.0 27 38.0 42.3 40.9 28 38.4 41.1 39.929 37.0 38.5 37.2 30 35.0 37.0 36.0 Correlation coefficient — 0.9970.997 Slope — 0.963 0.988 Intercept — 4.7 2.7

The results measured by using the second reagent containing the organicsilicon compound of the present invention exhibited improved slope andintercept as compared with the results measured by using the secondreagent not containing the organic silicon compound of the presentinvention. This indicates that the organic silicon compound of thepresent invention established specific conditions under which thereaction between cholesterols in HDL and enzymes is predominant over thereaction between cholesterols in LDL and enzymes, whereby thecharacteristics of the surfactant having lipoprotein selectivity wasfurther improved.

Example 5

The effect of the present invention was confirmed in the same conditionas in Example 2 except that 20 serum samples containing lipoproteinswere used as the samples and the organic silicon compounds shown inTable 5 were used in the second reagent. In Table 5, the organic siliconcompounds of Examples 5A-5D were used for formulating the second reagentafter dissolving these compounds in ethanol to a concentration of 10%.The organic silicon compounds of Examples 5E and 5F were used forformulating the second reagent after dissolving and mixing thesecompounds in ethanol together with SH8400 (modified silicone oilmanufactured by Dow Corning Toray Silicone Co., Ltd.) to a respectiveconcentration of 10%. The results are shown in Table 6.

TABLE 5 Example Organic silicon compound* 5A BY11-030(0.03%) 5BBY22-008M(0.05%) 5C SH-244(0.03%) 5D SH-245(0.05%) 5E DC345(0.02%) 5FSH200C-2cs(0.02%) *All manufactured by Dow Corning Toray Silicone Co.,Ltd.

TABLE 6 Unit: mg/dL Precipitation Method of the present invention Samplemethod Control Example 5A Example 5B Example 5C Example 5D Example 5EExample 5F 1 90.4 92.0 93.0 94.5 93.2 94.7 93.0 92.7 2 81.4 83.1 83.184.3 82.8 82.8 85.9 83.5 3 77.9 81.5 82.6 82.0 82.2 82.6 82.8 82.9 474.4 77.9 78.2 79.2 78.5 79.2 78.5 78.0 5 72.0 72.9 74.0 76.0 73.1 74.275.0 74.2 6 69.9 70.8 71.1 71.4 71.1 71.9 71.4 70.7 7 68.0 67.2 68.869.3 68.6 69.2 69.6 68.4 8 65.4 70.4 70.3 71.0 69.6 71.8 70.5 69.9 965.3 66.7 66.8 67.1 66.1 67.9 68.2 67.2 10 59.9 62.8 62.4 63.3 62.7 63.662.6 62.0 11 58.7 60.7 60.6 60.9 61.3 61.7 60.7 60.4 12 57.9 61.2 61.662.1 61.2 61.6 62.0 61.6 13 56.2 58.5 57.1 57.8 57.6 59.1 58.6 57.4 1454.0 55.6 55.6 57.3 56.3 55.7 56.7 55.6 15 52.7 55.3 55.4 55.5 54.9 55.755.6 55.3 16 51.8 52.1 52.4 52.7 52.6 53.2 53.1 52.4 17 50.0 51.1 52.152.4 50.5 52.0 51.7 52.1 18 49.4 52.3 52.5 51.9 51.7 52.3 52.0 52.3 1943.0 46.1 44.7 44.9 45.5 45.4 44.8 44.2 20 41.1 43.9 43.2 44.0 43.4 43.743.8 43.5 Correlation — 0.995 0.996 0.996 0.996 0.996 0.997 0.996coefficient Slope — 0.989 1.021 1.038 1.017 1.029 1.037 1.023 Intercept— 2.81 0.99 0.54 1.15 1.15 0.56 0.82

The results measured by using the second reagent containing the organicsilicon compound of the present invention exhibited particularlyimproved intercept as compared with the results measured by using thesecond reagent not containing the organic silicon compound of thepresent invention. This indicates that the organic silicon compound ofthe present invention established specific conditions under which thereaction with cholesterols in HDL is predominant over the reactionbetween cholesterols in LDL and enzymes, whereby the characteristics ofthe surfactant having lipoprotein selectivity was further improved.

INDUSTRIAL APPLICABILITY

Lipids in specific fractions (e.g. cholesterols) can be quantitativelydetermined efficiently by a simple procedure without requiring apretreatment such as centrifugation by using the method of the presentinvention. In addition, because the method allows specific determinationby a simple method using a small amount of sample, the method can beapplied to various types of automatic analyzers. The method is thusextremely useful in the field of clinical diagnosis. The highlipoprotein selectivity particularly improves the intercept and slope,providing an advantage of minute lipoprotein selectivity control.

1-16. (canceled)
 17. A fractionation method of assaying lipid componentsin a specific lipoprotein fraction, comprising assaying the lipid inblood components in the presence of an organic silicon compound.
 18. Themethod according to claim 17, wherein the organic silicon compound is asilicone, a derivative of silicone or a combination thereof.
 19. Themethod according to claim 18, wherein the silicone or the derivative ofsilicone is at least one member selected from the group consisting ofhigh polymerization silicone, cyclic silicone, silicone-containingsurfactant, and modified silicone oil.
 20. The method according to claim17, wherein the lipid component is a cholesterol, triglycerides, orphospholipid.
 21. The method according to claim 17, wherein a lipidcontained in the lipoprotein is detected by contacting the lipoproteinwith an enzyme, under conditions in which the organic silicon compoundis present and in the absence of a precipitation agent.
 22. The methodaccording to claim 21, wherein the contacting step is performed in thepresence of a surfactant having lipoprotein selectivity.
 23. A reagentfor assaying lipid components in a specific lipoprotein fraction,comprising an organic silicon compound.
 24. The reagent according toclaim 23, wherein the organic silicon compound is a silicone, aderivative of silicone or a combination thereof.
 25. The reagentaccording to claim 24, wherein the silicone or the derivative ofsilicone is at least one member selected from the group consisting ofhigh polymerization silicone, cyclic silicone, silicone-containingsurfactant, and modified silicone oil.
 26. The reagent according toclaim 23, wherein the lipid component is a cholesterol, triglycerides,or phospholipid.